Stroke remains the second leading cause of death worldwide and the primary cause of long-term disability in the US. Although pre-clinical studies have identified hundreds of potential drug agents, the only pharmacological treatment available remains early reperfusion with thrombolytics. A major factor for translational failure may be the overwhelming use of young male rodents in pre-clinical research, and lack of sex-specific design and analysis in clinical trials. Stroke is a sexually dimorphic disease with sex differences in incidence, prevalence, and outcome. Mitochondria mediate post-ischemia cell death in both males and females, but in different manners. Non-coding RNAs, including microRNAs (miRs), are upstream regulators of genes that regulate cell survival and mitochondrial function. In young adult male animals, miRs have been established as central regulators in the cellular response to stroke, however their role in females or aged animal cohorts represents a critical knowledge gap. Our prior studies (2, 4) and preliminary evidence suggest age-related differences between females and males in expression of select miRs and their targets following stroke. The overarching hypothesis of these Aims is that expression and function of miR-181a and miR-200c is a critical determinant of sexual dimorphism in stroke outcomes. To test this, we will first compare aged (20 month old) female and male mice using the middle cerebral artery occlusion (MCAO) model of experimental stroke, then compare primary female and male neuronal and astrocyte cultures utilizing in vitro ischemia. Preliminary evidence in aged animals reveals only a modest post- MCAO miR-181a response that is limited to females, while comparatively, a pronounced miR-200c response occurs in both sexes. Moreover, our preliminary evidence implies sex-differences in miR-181a and miR-200c gene targeting. In Aim 1, we will first compare protection with post-MCAO IV treatment of anti-miR-181a or anti- miR-200c in aged males and females. Then, we will assess the long-term cell-specific and sex-specific response in miR-181a and miR-200c to MCAO. Next, we will compare sustained post-MCAO miR-200c inhibition on long- term neuro-recovery between aged males and females, with the basis that preliminary evidence implies female- specific targeting of the neurotrophic protein reelin by miR-200c. In Aim 2, we will: 1) focus on the roles of miR- 181a and miR-200c in differences in regional (core versus penumbra) post-stroke disruption in mitochondrial function between aged males and females; and, 2) determine the roles of miR-181a and miR-200c in cell-type, and sex-specific alterations in oxidative phosphorylation and disruptions in cytosolic and mitochondrial Ca2+- handling following in vitro ischemia. In Aim 3 we will first define cell-type and sex-dependent differential gene targeting by miR-181a (Grp78 and XIAP) and miR-200c (XIAP, sirtuin-1, reelin). Finally we will determine whether observed differential gene targeting could be attributed to differences in RNA methylation, and whether this changes in response to injury. Delineating the molecular mechanisms that determine sexual dimorphism in stroke is necessary to overcome translational barriers and advance the development of novel stroke treatments.